Difference between revisions of "Part:BBa K3776002"
 
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===Characterization from iGEM23-NJTech-China-B===
 
===Characterization from iGEM23-NJTech-China-B===
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    <div class="h3">DavA</div>
 
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        5-Aminovalerateis produced from L-lysine through the 5-aminovalerate pathway.  5-aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. In our experiment, DavA catalyzes 5-aminovaleramide into 5-aminovalerate.                           
 
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      <div class="h3">1. Construct design</div>
 
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        <b>1.1 The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)</b>
 
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        The gene of DavA was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavA. By sequencing, the correct plasmid pRSFDuet-DavA was then transformed to E. coli BL21(DE3) (Figure 1). The gene of DavB was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavB. By sequencing, the correct plasmid pRSFDuet-DavB was then transformed to E. coli BL21(DE3) (Figure 1).
 
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      <p class="photo-desc">Figure1The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)</p>
 
  
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===DavA===
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         <b>1.2 The colony PCR of pRSFDuet-DavA in E. coli BL21</b>(DE3)                        
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5-Aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. Here we characterized the length of the davA gene, the molecular weight of DavA protein and the activity of DavA catalytic production of 5-aminovalerate for future iGEM teams.<html lang="en">
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         The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavA was successful obtained (Figure 2).                             </p>
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    <h2>1 Construct design</h2>
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    <h3>1.1 The transformation of palsmid pRSFDuet-davA into <i>Escherichia coli</i> BL21(DE3)</h3>
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          <img class="earth rotatehidden" src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution32.png">
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        The gene of davA was incorporated into plasmid pRSFDuet to obtain the plasmid pRSFDuet-davA. By sequencing, the correct plasmid pRSFDuet-davA was then transformed to <i>E. coli</i> BL21(DE3) (Figure 1). <br>
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         <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1211.png" width="45%" height="45%"></p>
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      <div class="h3">2.Protein expression of DavA</div>
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        The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. We transformed the plasmid pRSFDuet without DavA gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 3).                           </p>
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        <strong>Figure 1. The transformation of palsmid pRSFDuet-davA into <i>E. coli</i> BL21(DE3)</strong>
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    <h3>1.2 The colony PCR of pRSFDuet-davA in <i>E. coli</i> BL21(DE3)</h3>  
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         The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-davA was successful obtained (Figure 2).<br>
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    </p>
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    <div align="center">
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         <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1212.png" width="40%" height="45%"></p>
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        <strong>Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-davA</strong>
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        M represents the band of DNA marker; Lane 1, 2 and 3 represent the band of different colonies containing plasmid pRSFDuet-davA
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    </p>
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    <h2>2 Protein expression of DavA</h2>
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    <p>
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      The correct colony of recombinant <i>E. coli</i> BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We transformed the plasmid pRSFDuet without davA gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 3).
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    </p>
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    <div align="center">
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         <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution122.png" width="40%" height="45%"></p>
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    <div align="center">
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        <strong>Figure3. SDS-PAGE analysis of DavA expression in <i>E. coli</i> BL21(DE3)</strong>
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    <p>
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        Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of <i>E. coli</i> BL21(DE3) containing pRSFDuet; lanes 3 and 4:supernatant and precipitation of <i>E. coli</i> BL21(DE3) containing PRSFDuet-davA
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    <h2>3 Determination of DavB activity</h2>
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    <h3>3.1 Determination of DavA whole-cells catalytic activity</h3>
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    <p>
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        DavA catalyzes 5-aminovaleramide into 5-aminovalerate. We tested its activity by detecting production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 4).
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1231.png" width="45%" height="45%"></p>
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        <strong>Figure 4 The enzymatic activity of whole-cells congtaining DavA</strong>
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    <h3>3.2 Detemination of crude enzyme of DavA</h3>
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    <p>
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        The correct colony of recombinant <i>E. coli</i> BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We tested its activity by detecting production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 5).
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    </p>
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1232.png" width="45%" height="45%"></p>
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        <strong>Figure 5. The enzymatic activity of crude enzyme of DavA</strong>
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      <p class="photo-desc">Figure3. SDS-PAGE analysis of DavA expression in E. coli BL21(DE3)</p>
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      <p class="photo-desc">Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant  </p>
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      <p class="photo-desc">and precipitation of E. coli BL21(DE3) containg PRSFDuet-DavA</p>
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      <div class="h3">3. Determination of DavA activity</div>
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      <p class="content-paragraph">
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        <b>3.1 Determination of DavA whole-cells catalytic activity</b>
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      </p>
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      <p class="content-paragraph">
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        DavA catalyzes 5-aminovaleramide into 5- aminovalerate.The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37 ℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. Subsequently, we tested its activity by detecting production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 4).                           
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      <p class="photo-desc">Figure4 The enzymatic activity of whole-cells</p>
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        <b>3.2 Detemination of crude enzyme of DavA </b>
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        The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. Subsequently, we tested its activity by detecting production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 5).                         
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      <p class="photo-desc">Figure5 The enzymatic activity of crude enzyme</p>
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Latest revision as of 10:44, 12 October 2023


DavA

DavA, 5-aminovaleramide amidohydrolase, catalyzes the reaction of 5-aminovaleramide to 5-aminovalerate in the natural 5-aminovalerate pathway in Pseudomonas putida KT2440. This DavA sequence has been codon optimized for expression in Synechococcus elongatus UTEX 2973.

References

Liu, P., Zhang, H., Lv, M., Hu, M., Li, Z., Gao, C., Xu, P., & Ma, C. (2014). Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase. Scientific Reports, 4(1). https://doi.org/10.1038/srep05657

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution From NJTech-China-B 2023

Group:iGEM NJTech-China-B

Author: Yao Yao

Characterization from iGEM23-NJTech-China-B

DavA

5-Aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. Here we characterized the length of the davA gene, the molecular weight of DavA protein and the activity of DavA catalytic production of 5-aminovalerate for future iGEM teams.

1 Construct design

1.1 The transformation of palsmid pRSFDuet-davA into Escherichia coli BL21(DE3)

The gene of davA was incorporated into plasmid pRSFDuet to obtain the plasmid pRSFDuet-davA. By sequencing, the correct plasmid pRSFDuet-davA was then transformed to E. coli BL21(DE3) (Figure 1).

Figure 1. The transformation of palsmid pRSFDuet-davA into E. coli BL21(DE3)

1.2 The colony PCR of pRSFDuet-davA in E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-davA was successful obtained (Figure 2).

Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-davA

M represents the band of DNA marker; Lane 1, 2 and 3 represent the band of different colonies containing plasmid pRSFDuet-davA

2 Protein expression of DavA

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We transformed the plasmid pRSFDuet without davA gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 3).

Figure3. SDS-PAGE analysis of DavA expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet; lanes 3 and 4:supernatant and precipitation of E. coli BL21(DE3) containing PRSFDuet-davA

3 Determination of DavB activity

3.1 Determination of DavA whole-cells catalytic activity

DavA catalyzes 5-aminovaleramide into 5-aminovalerate. We tested its activity by detecting production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 4).

Figure 4 The enzymatic activity of whole-cells congtaining DavA

3.2 Detemination of crude enzyme of DavA

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We tested its activity by detecting production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 5).

Figure 5. The enzymatic activity of crude enzyme of DavA